Session III – Homologous Replacement/Gene Targeting

Optimising gene repair strategies in cell culture

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Gene repair, the precise modification of the genome, offers a number of advantages over replacement gene therapy. In practice, gene targeting strategies are limited by the inefficiency of homologous recombination in mammalian cells. A number of strategies, including RNA-DNA oligonucleotides (RDOs) and short DNA fragments (SDFs), show promise in improving the efficiency of gene correction. We are using GFP as a reporter for gene repair in living cells. A single base substitution was introduced into GFP to create a nonsense mutation (STOP codon, W399X). RDOs and SDFs are used to repair this mutation episomally in transient transfections and restore green fluorescence. The correction efficiency is determined by FACS analysis. SDFs appear to correct GFP W399X in a number of different cell lines (COS7, A549, HT1080, HuH-7), although all at a similar low frequency (0.6% of transfected cells). RDOs correct only one of our cell lines significantly (HT1080-RAD51), these cells overexpress the human RAD51 gene; the bacterial RecA homologue. The GFP W399X reporter is a fusion gene with hygromycin (at the 5′ end), this has allowed us to make stable cell lines (A549, HT1080) to study genomic correction. Initial studies using our correction molecules show only low efficiencies of genomic repair (10−4). Polyethylenimine (PEI) is used to deliver RDOs and SDFs into mammalian cells in culture for our study. We have used fluorescently labelled RDOs and SDFs to study the effectiveness of this process. FACS analysis of transfected nuclei implied efficient delivery (>90%) both with SDFs and RDOs. However, confocal fluorescence microscopy suggests that a large proportion of the complexed RDO/SDF appears to remain outside the nucleus (or attached to the nuclear membrane). On the basis of these data we are assessing new delivery methods and factors that may alter recombination status to optimise gene repair.


Cystic fibrosis (CF) is a monogenic disease affecting multiple organs. Lung pathology, typified by thick viscous mucous and susceptibility to chronic bacterial infection, is important in determining clinical decline of CF patients. This organ is readily accessible to inhaled medication and consequently antibiotic and mucolytic agents help to abate the disease and increase life expectancy. Both the monogenic cause of CF and the lung's accessibility have made CF a focus of gene therapy for over 10 years. Unfortunately, attempts to replace the defective gene responsible for CF, the CF transmembrane conductance regulator (CFTR) gene with a functional copy have failed to benefit patients in the clinic. The reasons for this are numerous and include immune defences against viral delivery, inefficient non-viral gene delivery, transgene loss and transcriptional silencing. This has motivated clinical researchers to devise new and elaborate ways to overcome these barriers.1234

In contrast to gene replacement, correcting mutations within the endogenous gene offers a more elegant approach to genetic medicine. Precisely repairing a genetic lesion would preserve the regulatory and splicing machinery of a gene. However, this strategy suffers from two weaknesses: (1) inefficient homologous recombination in mammalian cells; and (2) random and potentially mutagenic genomic integration of the targeting construct.5 Nevertheless, a number of strategies are currently being explored to overcome these barriers (for a recent review see Richardson et al).6

Our work seeks to exploit these new technologies to target CFTR mutations in lung epithelial cells (or lung progenitor cells). We have focused our efforts on two strategies: (1) short fragment homologous recombination (SFHR) and (2) chimeraplasty.

(1) SFHR utilises the observation that short DNA fragments (SDFs) of approximately 500 bp, which are identical in sequence to their target (with the exception of a centrally located mismatch) induce gene repair in mammalian cells.7 Molecular evidence has recently been presented that this strategy works in cell culture for CF8 and in vivo in mice.910

(2) RNA-DNA oligonucleotides (RDOs, or chimeraplasts) are chimeric oligonucleotides, which consist of both DNA and 2’-O-methyl RNA residues. These fold to form a double-stranded sequence with polythymidine hairpin loops at each end, a central pentameric DNA sequence (containing a single mismatch from their target) and flanking sequences which are DNA:RNA heteroduplexes (see Figure 1). These molecules have been used extensively in cell free, cell culture and in vivo systems (refer to Richardson et al6 for a thorough review of this work). The initial RDO design (such as in Figure 1) has been modified in recent studies and the importance of certain structural features dissected.11121314

Figure 1

Gene correction reagents. SDFGFP, both primers sequences are shown for constructing this 442 bp linear fragment, which is used to correct GFP W399X. RDOGFP, the sequence (and folded structure) of this chimeric oligonucleotide is shown. The asterisk (*) denotes the position of the mismatch from the reporter construct.

Results and discussion

Ideally we wish to target the CFTR gene and demonstrate functional change that may extrapolate to potential clinical benefit. However, assays to detect functional CFTR protein in a background of mutant molecules are very difficult. Therefore to optimise gene correction, we created a reporter system that allows us to measure gene repair directly in living cells. Gene repair reporters have proved effective1112131516171819202122 and we hope that this system will allow application of this technology to living cells (see also Liu et al14). A single base substitution was introduced into the EGFP gene (from Clontech's pHygEGFP vector (Clontech, Palo Alto, CA, USA)) to create a premature stop codon (GFP W399X) which inactivates the green fluorescence of EGFP. This reporter system allows us to monitor the appearance of green fluorescence as a marker for gene repair. To correct this mutation a 442 bp SDFGFP was made by the PCR and a RDOGFP synthesised (25 bp of homology, Figure 1).

The GFP W399X reporter plasmid was used to study the gene repair activity of our gene correction molecules Figure 1within mammalian cells. Various cell lines (Table 1) were transfected with both plasmid (GFP W399X) and either SDFGFP or RDOGFP using a polyethylinimine (PEI) protocol (M Cotton, personal communication). After 48 h, the appearance of green cells was monitored by FACS analysis. We used a two-colour FACS analysis (dual wavelength differential fluorescence correction) to accurately and sensitively determine the number of green cells within a sample. The SDFGFP produced green cells across a range of cell lines, whereas RDOGFP only produced green cells in HT1080 cell line that overexpresses human RAD5122 (a gift from A Porter, MRC Clinical Science Centre, Imperial College School of Medicine, London, UK). The data are summarised in Table 1. The differences in correction efficiency between cell lines or between methods (SDF or RDO) are not statistically significant. Only HeLa cells failed to correct GFP W399X with both types of correction molecules. Encouragingly (from a CF point of view), A549 cells derived from a lung carcinoma show successful repair with SDFs.

Table1 The efficiency of episomal (GFP W399X) gene correction with SDFs and RDOs in a range of mammalian cell lines

Observing the repair of DNA sequences within plasmids which have been transiently transfected into cells is distinct from repair of the genome. Chromosomal and extrachromosomal repair mechanisms may differ. To address this, we have constructed stable cell lines utilising the hygromycin resistance component of our reporter system. The GFP W399X plasmid was linearised and transfected into cells, which were then subjected to hygromycin selection. Resulting colonies were isolated and the clones expanded and checked for reporter gene expression. Our initial results with these cell lines failed to demonstrate any gene correction using RDOGFP either in HT1080-GFPW399X cells or in HT1080-GFPW399X cells overexpressing RAD51. For SDF correction, we have made a number of different sized SDFGFPs (442 bp SDF442GFP; 600 bp SDF600GFP; 1600 bp SDF1600GFP). We have seen that the larger the SDFGFP the more efficient the gene repair (Table 2).

Table2 Preliminary results of gene correction of HT1080-GFPW399X and HT1080-GFPW399X-RAD51 cells

We wished to test whether the generally held notion that small molecules (such as oligonucleotides) can be efficiently delivered to the nucleus (but are less stable) was true in our culture system. In addition we were concerned that the observed variation in repair efficiency was related to variation in nuclear delivery of our correction molecules. We made fluorescently tagged versions of both SDF442GFP and RDOGFP (termed SDF* and RDO*). Confocal microscopic analysis at 24 and 48 h after transfection showed that the majority of the complex appeared to be outside the nucleus (or perhaps attached to the nuclear membrane23). Live cell imaging was performed with both SDF* and RDO* in HT1080 cells during transfection. This also demonstrated that PEI (and also lipid or lipid/peptide) transfections failed to deliver the bulk of the DNA into the nucleus. If the process of recombination is dependent upon the nuclear concentration of targeting molecule, then this delivery problem may be a limiting factor for repair in our system (see Lukacsovich et al24 for discussion). The observed pattern was the same in each of the cell lines used for the episomal correction study (Table 1).

To assess the stability of linear SDFs in cell culture, a 1.6 kb linear gene was generated by the PCR. This created a GFP gene under the control of a CMV promoter. After transfection into HT1080 cells, the number of green cells was monitored at different times. A 24-h pre-treatment of cells with trichostatin A (TSA) gave an indication of genomic integration (this histone deactylase inhibitor increases expression of some integrated transgenes: X Nan, personal communication). The results show that after an initial peak of green cells (~12% at 2 days after transfection), transgene expression reduces gradually (~3% at day 7). However, green cells were still observed up to 21 days after transfection (0.15%). At 28 days, the remaining green cells had become sensitive to TSA enhancement, indicative of transgene integration. These data imply that whilst the bulk of the SDFs and RDOs fail to enter the nucleus in our cell culture system, those that are within the nucleus have the potential to remain there for a considerable time.

A number of key safety problems need to be addressed before gene repair can proceed to clinical application. However, an exciting prospect which could bypass some of these hurdles is to exploit stem cells or progenitors cells. If stem cells can be isolated and corrected ex vivo, then re-implanted, a diseased organ could remain corrected for the lifetime of the patient. The successful use of stem cells in replacement gene therapy has already been demonstrated25 and pluripotent stem cells from bone marrow have been shown to give rise to lung epithelia in the mouse.26 We are investigating the possibility of using murine airway epithelial stem cell niches, such as those identified by Borthwick et al,27 as a source of cells for gene repair.


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Correspondence to P Thorpe.

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  • CFTR
  • SFHR
  • gene repair
  • chimeraplast

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